Stop-And-Go DNA Pyrosequencing

A set of modified nucleotides prepared by Jingyue Ju, Nicholas J. Turro, and coworkers at Columbia University could improve the accuracy of the DNA-sequencing technique known as pyrosequencing in homopolymeric regions of DNA where the same base is repeated (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.0707495104). In pyrosequencing, DNA is sequenced by detecting the chemiluminescence triggered by the release of pyrophosphate when a nucleotide is incorporated in the growing DNA strand. Pyrosequencing suffers in homopolymeric regions because the chemiluminescence signal is not exactly proportional to the number of incorporated bases, especially in stretches of more than five repeated bases. The Columbia team now reports nucleotide reversible terminators for pyrosequencing in which the 3′-hydroxyl group of the nucleotide is capped with a removable 2-nitrobenzyl or allyl group. The modified nucleotides temporarily stop the sequencing reaction until the capping moiety is removed, clearly separating the signal from the repeated nucleotides and leading to accurate identification of bases in the homopolymeric regions.